Current HIV-1 vaccine candidates based on recombinant adenovirus serotype 5 (rAd5) vectors are promising but may prove limited by the high prevalence of pre-existing anti-Ad5 immunity in the developing world. To overcome this problem, we have constructed a series of novel serotype and novel chimeric rAd vectors that effectively circumvent anti-Ad5 immunity. We have also demonstrated that heterologous rAd prime-boost regimens utilizing two serologically distinct rAd vectors elicit remarkably potent immune responses, particularly regimens involving rAd26 priming and rAd5 or rAd5HVR48 boosting. In addition, we have generated rAd vectors expressing optimal C clade, M consensus, and M mosaic HIV-1 antigens designed to minimize genetic distance and to optimize T cell epitope coverage among global HIV-1 sequences. We therefore propose to develop a practical, two-injection, heterologous rAd26 prime, rAd5HVR48 boost regimen expressing antigens optimized for global coverage as a novel candidate HIV-1 vaccine. The goals of Project 2 are to define the optimal rAd vector combination in phase 1 studies and to advance this novel HIV-1 vaccine candidate into expanded international phase 2a studies in collaboration with the HIV Vaccine Trials Network (HVTN). The clinical studies in Project 2 will build on the preclinical studies in Project 1 and the manufacturing program in Project 3. In Project 2, we hypothesize that the heterologous rAd26 prime, rAd5HVR48 boost regimen will prove safe and highly immunogenic in humans in both the United States and sub-Saharan Africa. We further hypothesize that this HIV-1 vaccine candidate will not be suppressed by pre-existing antivector immunity and will be a promising candidate for further development into advanced phase clinical trials by the end of this project period. To explore these hypotheses, we propose the following three Specific Aims: 1. To perform a phase 1 study assessing the safety and immunogenicity of rAd26 and rAd5HVR48 HIV-1 vaccine regimens in the United States; 2. To perform a phase 1 study assessing the safety and immunogenicity of rAd26 and rAd5HVR48 HIV-1 vaccine regimens in South Africa; and 3. To perform an international phase 2a study evaluating expanded safety and immunogenicity of the heterologous rAd26 prime, rAd5HVR48 boost HIV-1 vaccine regimen.